With the recent first draft completion of the human genome, much attention is now shifting to the field of proteomics, where gene products (proteins), their various variants, interacting partners and the dynamics of their regulation and processing are the emphasis of study. Such studies are essential in understanding, for example, the mechanisms behind genetic/environmentally induced disorders or the influences of drug mediated therapies, as well as potentially becoming the underlying foundation for further clinical and diagnostic analyses. Critical to these studies is the ability to qualitatively determine specific variants of whole proteins (i.e., splice variants, point mutations and posttranslationally modified versions) and the ability to view their quantitative modulation.
Traditionally, distinctly different assays are used in the qualitative analysis of proteins/variants and in monitoring their quantitative modulation. Qualitative analyses generally rely on structurally related techniques such as NMR or X-Ray crystallography, or, oftentimes, are accomplished on the gene level through gene sequencing or various methodologies of single nucleotide polymorphism analyses. Quantitative analyses rely, more so, on functional related properties of the proteinaceous analyte, and include various immunoassays (enzyme-linked immunosorbent assays and radioimmunoassays) or biosensor type instrumentation. As such, there is a lack in any one assay to qualitatively differentiate between different variants of a specific protein while still maintaining quantitative capabilities.
As an example, β-2-microglobulin (β2m) is a low molecular mass protein identified as the light chain of the Class I major histocompatibility complex synthesized in all nucleated cells. Upon activation of the immune system, both B- and T-lymphocytes actively release β2m into circulation where it is later eliminated via glomerular filtration and tubular reabsorption. Serum levels of β2m have been measured and nominally correlated to ailments such as AIDS, rheumatoid arthritis, leukemia, myeloma and malignant lymphoma. On the other hand, β2m levels in urine are indicators of glomerular filtration rate and tubular reabsorption. Conventionally, β2m levels are monitored using a variety of immuno-based assays, including enzyme-linked immunosorbent assays, radioimmunoassays and particle-enhanced turbidimetry assays. The quantitative dynamic range (spanning β2m concentrations of ˜0.2-20 mg/L) and the accuracy (1-10%) of the assays are sufficient to cover the normal and elevated levels of β2m in a variety of biological fluids. One drawback of these conventional assays, however, is the inability to differentiate between the wild type and variants of β2m. Such distinction is important when considering that genetic and posttranslational variants of β2m are indicative of ailments distinct from those indicated by the wild type protein. Several separate assays are thus required to discern between these different protein variants, and to date, no such combination of assays has been used in the study of β2m.
Thus, there exists a need for a single assay capable of assessing proteins present in a variety of biological fluids, both qualitatively and quantitatively. New and novel technologies are needed to fulfill these needs. Importantly, these technologies must: 1) be able to selectively retrieve and concentrate specific proteins/biomarkers from biological fluid for subsequent high-performance analyses, 2) be able to quantify targeted proteins, 3) be able to recognize variants of targeted proteins (e.g., splice variants, point mutations and posttranslational modifications) and to elucidate their nature, and 4) be capable of analyzing for, and identifying, ligands interacting with targeted proteins.
Two protein mass spectrometry techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization mass spectrometry, offer the particular advantage of differentiating between different mass-shifted forms of the same protein. In this manner, a single pan-antibody can be used to retrieve all protein variants from a biological fluid, upon which each variant is detected during mass spectrometry at a unique and characteristic molecular mass. Mass resolution of related species also allows mass-shifted variants of a target protein to be intentionally incorporated into the analysis for use as internal reference standards for quantitative analysis. This step forms the basis of a mass spectrometric immunoassay (MSIA), an assay that can be used for the unambiguous detection and rigorous quantification of polypeptides/proteins retrieved from complex biological systems.
For the foregoing reasons, there is a need for MSIA devices, methods and kits for the rapid and efficient analysis of specific proteins and variants present in various biological fluids.